NextSeq 500 sequencing; ChIP-seq of wild-type SOX18 and a dominant-negative variant (SOX18RaOp) in HeLa cells
ChIP-seq of wild-type SOX18 and a dominant-negative variant (SOX18RaOp) in HeLa cells
Hela cells were seeded in 3×15 cm dishes per condition at 2.65×106 cells/dish, and then transfected for 24 h with the following combination of plasmids: (1) 15 μg pcDNA3.1 glomyc-mSOX18 and 15 μg untagged mSOX18; (2) 15 μg pcDNA myc-RaOP and 15 μg untagged RaOP; (3) 15 μg pcDNA3.1 glomyc-mSOX18 and 15 μg untagged RaOP. Transfections were performed using the X-tremeGENE 9 Transfection Reagent kit (Roche) as per manufacturer's instructions, with a DNA to X-tremeGENE 9 ratio of 1 μg to 3 μL.Cells were then fixed in molecular biology grade formaldehyde solution in their existing media to a final concentration of 11% formaldehyde, 0.1 M NaCl, 1 mM EDTA, 50 mM HEPES for 15 min at room temperature with agitation. Fixing was quenched by adding a glycine solution to a final concentration of 2.5 M for 5 min at room temperature. Cells were then scraped and pelleted at 800 g for 10 min at 4°C. Pellets were washed with 10 mL ice-cold PBS, pooled, and then centrifuged at 800 g for 10 min at 4°C. Pellets were resuspended in 10 mL ice-cold PBS supplemented with cOmplete protease inhibitor (Roche), and then centrifuged at 800 g for 10 min at 4°C. Supernatant were removed completely, and the resultant pellets were snap-frozen in dry ice, and then stored at -80°C until analysis. Hela cells were grown in 10% FCS DMEM (supplemented with GlutaMAX) in monolayer at 37°C with 5% atmospheric CO2. Nucleic acid extraction was performed by Active Motif (Carlsbad, CA). Chromatin was isolated by adding lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300-500 bp with Active Motif's EpiShear probe sonicator (cat# 53051). Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by SPRI beads clean up (Beckman Coulter) and quantitation by Clariostar (BMG Labtech). Extrapolation to the original chromatin volume allowed determination of the total chromatin yield.  An aliquot of chromatin (40 ug) was precleared with protein G agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using 6 ug of antibody against Myc-tag (Abcam, ab9132, Lot# GR3325755-1). Complexes were eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65 oC, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation.  Nucleic acid library construction was performed by Active Motif (Carlsbad, CA). Illumina sequencing libraries were prepared from the ChIP and Input DNAs by the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation. Steps were performed on an automated system (Apollo 342, Wafergen Biosystems/Takara).